US9617315B2 - Artificial polypeptide fiber and method for producing the same - Google Patents

Artificial polypeptide fiber and method for producing the same Download PDF

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US9617315B2
US9617315B2 US14/114,636 US201214114636A US9617315B2 US 9617315 B2 US9617315 B2 US 9617315B2 US 201214114636 A US201214114636 A US 201214114636A US 9617315 B2 US9617315 B2 US 9617315B2
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fiber
polypeptide
amino acid
producing
artificial polypeptide
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US20140058066A1 (en
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Kazuhide Sekiyama
Kaori Sekiyama
Mizuki Ishikawa
Ryota Sato
Shinya Murata
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Spiber Inc
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Spiber Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/06—Wet spinning methods
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/12—Stretch-spinning methods
    • D01D5/16—Stretch-spinning methods using rollers, or like mechanical devices, e.g. snubbing pins
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D01F4/02—Monocomponent artificial filaments or the like of proteins; Manufacture thereof from fibroin
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F6/00—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof
    • D01F6/58—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products
    • D01F6/68—Monocomponent artificial filaments or the like of synthetic polymers; Manufacture thereof from homopolycondensation products from polyaminoacids or polypeptides

Definitions

  • the present invention relates to an artificial polypeptide fiber that is one kind of synthetic protein fibers and a method for producing the same.
  • Spider silk fibers are fibers having high strength and toughness, and are known to have higher strength and toughness than high-tensile steels, nylon 6 fibers, aramid fibers, carbon fibers, etc. In addition, they have an advantage in that oil is not used as a raw material and biomass can be used instead.
  • Some artificial spider silk fibers also have been proposed.
  • Patent Document 1 has proposed a fiber produced by extruding a synthetic protein spinning solution into a coagulation bath with 90% methanol from a spinneret at a speed of 5 to 10 ⁇ l/min.
  • Patent Document 2 has proposed a fiber 1 ⁇ m or more in diameter and 5 mm or more in length having a tensile strength of 200 MPa or higher.
  • Non-Patent Document 1 has disclosed a drawn yarn having a strength of 1.91 to 2.26 g/d and an elongation of 43.4 to 59.6% obtained by drawing a yarn at a draw ratio of 5 times in a methanol bath and a water bath.
  • Non-Patent Document 2 has disclosed a drawn yarn having a stress of 600 MPa and an elongation of 25% obtained by drawing a yarn at a draw ratio of 5 times.
  • Non-Patent Document 3 has disclosed a drawn yarn having a stress of 280 to 350 MPa and an elongation of 30 to 40% obtained by keeping a yarn in steam for 5 minutes so as to draw the yarn to 5 times.
  • Patent Document 1 JP 2004-503204 A
  • Patent Document 2 JP 2009-521921 A
  • Non-Patent Document 1 Anthoula Lazaris, et. al., “Spider Silk Fiber Spun from Soluble Recombinant Silk Produced in Mammalian Cells”, Science, 295, page 472, Jan. 18, 2002
  • Non-Patent Document 2 Xiao-Xia Xia, et. al., “Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber”, PNAS, vol. 107, No. 32, pages 14059-14063, Aug. 10, 2010
  • Non-Patent Document 3 M. Elice, et. al., “Bioinspired Fibers follow the Track of Natural Spider Silk”, Macromolecules, Vol. 44, No. 5, pages 1166-1176, Apr. 2, 2011
  • the present invention provides an artificial polypeptide fiber having high stress and toughness, and a method for producing the same.
  • An artificial polypeptide fiber of the present invention is an artificial fiber containing a polypeptide as a main component, and has a stress of 350 MPa or more and a toughness of 138 MJ/m 3 or more.
  • a method for producing an artificial polypeptide fiber of the present invention is a method for producing an artificial polypeptide fiber that is obtained by spinning a spinning solution containing a polypeptide derived from natural spider silk proteins and performing drawing of at least two stages.
  • the drawing of at least two stages includes a first-stage drawing in wet heat and a second-stage drawing in dry heat.
  • the present invention can provide an artificial polypeptide fiber having high stress and toughness by drawing an undrawn fiber made of artificial polypeptide through at least two stages in wet heat and dry heat, and a method for producing the same. Specifically, it is possible to realize an artificial polypeptide fiber having a stress of 350 MPa or more and a toughness of 138 MJ/m 3 or more.
  • the fiber having high stress and high toughness is advantageous as a composite material formed with metal, resin, rubber, etc.
  • FIG. 1 illustrates a production process in one example of the present invention.
  • FIGS. 2A and 2B illustrate a production process in another example of the present invention.
  • FIG. 2A shows a spinning process and a first-stage drawing process
  • FIG. 2B shows a second-stage drawing process.
  • FIG. 3 shows a stress-displacement (strain) curve of a fiber obtained in Example 1 of the present invention.
  • FIG. 4 shows a stress-displacement (strain) curve of a fiber obtained in Example 2 of the present invention.
  • FIG. 5 shows a stress-displacement (strain) curve of a fiber obtained in Example 3 of the present invention.
  • FIG. 6 shows a stress-displacement (strain) curve of a fiber obtained in Example 4 of the present invention.
  • FIG. 7 shows a stress-displacement (strain) curve of a fiber obtained in Example 5 of the present invention.
  • FIG. 8 shows a stress-displacement (strain) curve of a fiber obtained in Comparative Example 1.
  • FIG. 9 shows a stress-displacement (strain) curve of a fiber obtained in Comparative Example 2.
  • an artificial polypeptide fiber of the present invention contains a polypeptide as a main component.
  • the term “main component” refers to a component contained in an amount of 80% by mass (mass %) or more, more preferably contained in an amount of 90 mass % or more, and further preferably contained in an amount of 95 mass % or more.
  • the artificial polypeptide fiber of the present invention may contain components other than polypeptides within a range that do not inhibit the effect of the present invention.
  • the polypeptide is not limited particularly insofar as it is derived from natural spider silk proteins.
  • the polypeptide preferably is derived from spigot dragline proteins produced in the major ampullate glands of spiders.
  • the spigot dragline proteins include major ampullate spidroin MaSp1 and MaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived from Araneus diadematus, etc.
  • Examples of the polypeptide derived from the spigot dragline proteins include variants, analogs, derivatives or the like of the spigot dragline proteins.
  • polypeptide derived from the spigot dragline proteins examples include a polypeptide containing two or more units of an amino acid sequence represented by the formula 1: REP1 ⁇ REP2 (1), preferably a polypeptide containing five or more units thereof, and more preferably a polypeptide containing ten or more units thereof.
  • the polypeptide derived from the spigot dragline proteins may be a polypeptide that contains units of the amino acid sequence represented by the formula 1: REP1 ⁇ REP2 (1) and that has, at a C-terminal, an amino acid sequence represented by any of SEQ ID NOS: 1 to 3 or an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by any of SEQ ID NOS: 1 to 3.
  • units of the amino acid sequence represented by the formula (1): REP1 ⁇ REP2 (1) may be the same or may be different from each other.
  • the units of the amino acid sequence represented by [REP1 ⁇ REP2 (1)] 1 being different from each other includes cases where the REP1 is different, the REP2 is different, and both of the REP1 and REP2 are different.
  • the molecular weight is preferably 300 kDa or less, more preferably 200 kDa or less, and further preferably 150 kDa or less, in view of productivity.
  • the REP1 is an amino acid sequence composed of 2 to 20 amino acid residues arranged in series that are selected from at least one of alanine and glycine, more preferably 3 to 16 amino acid residues arranged in series selected therefrom, further preferably 4 to 12 amino acid residues arranged in series selected therefrom, and most preferably 5 to 8 amino acid residues arranged in series selected therefrom.
  • the REP2 is an amino acid sequence composed of 2 to 200 amino acid residues, more preferably 10 to 150 amino acid residues, further preferably 20 to 100 amino acid residues and most preferably 20 to 75 amino acid residues, and the total number of glycine, serine, glutamine and alanine residues contained in the amino acid sequence is 40% or more, preferably 60% or more, and more preferably 70% or more with respect to the total number of amino acid residues contained therein.
  • the above REP1 corresponds to a crystal region in a fiber where a crystal ⁇ sheet is formed
  • the above REP2 corresponds to an amorphous region in a fiber where most of parts lack regular configurations and that has more flexibility.
  • the above [REP1 ⁇ REP2] corresponds to a repetitious region (repetitive sequence) composed of the crystal region and the amorphous region, which is a characteristic sequence of dragline proteins.
  • An amino acid sequence represented by SEQ ID NO: 1 is identical to an amino acid sequence that is composed of 50 amino acid residues of an amino acid sequence of ADF3 (GI: 1263287, NCBI) from the C-terminal.
  • An amino acid sequence represented by SEQ ID NO: 2 is identical to an amino acid sequence represented by SEQ ID NO: 1 in which 20 amino acid residues have been removed from the C-terminal.
  • An amino acid sequence represented by SEQ ID NO: 3 is identical to an amino acid sequence represented by SEQ ID NO: 1 in which 29 amino acid residues have been removed from the C-terminal.
  • polypeptide that contains units of the amino acid sequence represented by the formula 1: REP1 ⁇ REP2 (1) and that has, at a C-terminal, an amino acid sequence represented by any of SEQ ID NOS: 1 to 3 or an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by any of SEQ ID NOS: 1 to 3 is a polypeptide having an amino acid sequence represented by SEQ ID NO: 4.
  • the polypeptide having an amino acid sequence represented by SEQ ID NO: 4 is one obtained by the following mutation: in an amino acid sequence of ADF3 to which an amino acid sequence (SEQ ID NO: 7) composed of a start codon at a N-terminal, His 10 tags and an HRV3C Protease (Human rhinovirus 3C Protease) recognition site has been added, 1 st to 13 th repetitive regions are about doubled and the translation ends at the 1154 th amino acid residue.
  • the C-terminal sequence is identical to the amino acid sequence represented by SEQ ID NO: 3.
  • polypeptide that contains units of the amino acid sequence represented by the formula 1: REP1 ⁇ REP2 (1) and that has, at a C-terminal, an amino acid sequence represented by any of SEQ ID NOS: 1 to 3 or an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by any of SEQ ID NOS: 1 to 3 is a protein that has an amino acid sequence represented by SEQ ID NO: 4 in which one or a plurality of amino acids have been substituted, deleted, inserted and/or added and that has the repetitious region composed of the crystal region and the amorphous region.
  • “one or a plurality of” refers to 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 or a few, for example. Further, in the present invention, “one or a few” refers to 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.
  • the polypeptide can be produced using a host that has been transformed by an expression vector containing a gene encoding a polypeptide.
  • a method for producing a gene is not limited particularly, and it may be produced by amplifying a gene encoding a natural spider silk protein from a cell derived from spiders by a polymerase chain reaction (PCR) or the like for cloning, or may be synthesized chemically.
  • PCR polymerase chain reaction
  • a method for chemically synthesizing a gene also is not limited particularly, and it can be synthesized as follows, for example: based on information of amino acid sequences of natural spider silk proteins obtained from the NCBI web database, etc., oligonucleotides that have been synthesized automatically with AKTA oligopilot plus 10/100 (GE Healthcare Japan Corporation) are linked by PCR or the like. At this time, in order to facilitate the purification and observation of protein, it is possible to synthesize a gene that encodes a protein having an amino acid sequence of the above-described amino acid sequence to which an amino acid sequence composed of a start codon and His 10 tags has been added, to the N-terminal.
  • the expression vector examples include a plasmid, a phage, a virus, etc., that can express protein from a DNA sequence.
  • the plasmid type expression vector is not limited particularly insofar as it allows expression of a target gene in a host cell and it can amplify itself.
  • a pET22b(+) plasmid vector, a pCold plasmid vector and the like can be used.
  • the host include animal cells, plant cells, microbes, etc.
  • a spinning solution is prepared by adding a solvent to the above-described polypeptide and adjusting the viscosity to allow spinning.
  • the solvent is not limited particularly insofar as it can dissolve the polypeptide.
  • a solution containing hexafluoroisopropanol (HFIP), hexafluoroacetone (HFA), formic acid, urea, guanidine, sodium dodecyl sulfate (SDS), lithium bromide, calcium chloride, lithium thiocyanate or the like is used as a solvent and the solvent is added to the polypeptide in an appropriate amount so that the viscosity of the solution becomes 100 to 10,000 cP (centipoises). This is defined as the spinning solution.
  • a coagulation liquid to be used for the wet spinning is not limited particularly insofar as it allows desolvation.
  • the solvent is HFIP
  • the coagulation liquid preferably is a lower alcohol with a carbon number of 1 to 5 such as methanol, ethanol and 2-propanol, or acetone.
  • the temperature of the coagulation liquid preferably is 0-30° C. This range stabilizes spinning.
  • the extrusion speed preferably is set at 0.2-2.4 ml/h per one hole. This range stabilizes spinning.
  • the further preferable extrusion speed is 0.6-2.2 ml/h per one hole.
  • a length of a coagulation liquid bath is 200-500 mm
  • a take-up speed of the undrawn yarn is 1-3 m/min
  • residence time is 0.01-0.15 min.
  • Drawing is performed in at least two stages. Of course, it also is possible to adopt multistage drawing with three or more stages.
  • the reason for adopting the multistage drawing of two or more stages is as follows. Since molecules of polypeptides derived from natural spider silk proteins are less likely to be oriented, the multistage drawing is performed so as to orient the molecules stepwise and increase the total draw ratio. Consequently, high-toughness fibers can be obtained.
  • the drawing of at least two stages includes a first-stage drawing in wet heat and a second-stage drawing in dry heat.
  • the first-stage drawing in wet heat may be performed in the above-described coagulation liquid.
  • the wet heat of the first-stage drawing may be performed in hot water or by steam heating. An organic solvent, etc., may be added to the hot water.
  • the multistage drawing may include a drawing method other than the wet heat or dry heat in any stage of the drawing process.
  • the first-stage drawing it is preferable to draw an undrawn yarn to 2 to 8 times in hot water at 50-90° C. This allows stable drawing.
  • the second-stage drawing it is preferable to draw a yarn to 1.25 to 3 times in dry heat at 170-270° C. This provides a high-toughness drawn yarn.
  • the first-stage drawing described above it is further preferable to draw an undrawn yarn in hot water at 75-85° C.
  • the draw ratio of the first-stage drawing further preferably is to 2.3 to 7 times.
  • the dry heat further preferably is set at 180-230° C.
  • the draw ratio of the second-stage drawing further preferably is to 1.35 to 3 times.
  • the total draw ratio is preferably to more than 5 times and 20 times or less, and further preferably to 6 times or more and 11 times or less.
  • the dry heat is performed using an electric tubular furnace or a heat plate.
  • FIG. 1 illustrates a production process in one example of the present invention.
  • FIG. 1 shows a continuous process.
  • a spinning-drawing apparatus 10 includes an extrusion process 1 , an undrawn-yarn production process 2 , a wet-heat drawing process 3 , and a dry-heat drawing process 4 .
  • a spinning solution 6 is stored in a storage tank 7 and extruded from a gear pump 8 to a spinneret 9 .
  • a spinning solution may be filled in a cylinder and extruded from a nozzle using a syringe pump.
  • the extruded spinning solution is supplied directly or via an air gap 19 into a coagulation liquid 11 of a coagulation liquid bath 20 , and thus a solvent is removed.
  • an obtained yarn is supplied into hot water 12 in a drawing bath 21 and subjected to the first-stage drawing.
  • the drawing depends on a speed ratio between a supply nip roller 13 and a take-up nip roller 14 .
  • the yarn is supplied to a dry-heat drawing machine 17 and subjected to the second-stage drawing inside a guide 22 , whereby a yarn roll 5 is obtained.
  • the drawing depends on a speed ratio between a supply nip roller 15 and a take-up nip roller 16 .
  • 18 a to 18 f are yarn guides.
  • FIGS. 2A and 2B illustrate a case in which the production process is separated in another example of the present invention.
  • FIG. 2A shows a spinning process 30 and a first-stage drawing process 40
  • FIG. 2B shows a second-stage drawing process 50 .
  • a yarn may be wound or may be stored in a container without being wound.
  • a spinning solution 32 is contained in a microsyringe 31 and moved in a direction indicated by an arrow P using a syringe pump, so that the spinning solution 32 is extruded from a nozzle 33 and supplied into a coagulation liquid 35 in a coagulation liquid bath 34 .
  • an undrawn yarn 36 is obtained.
  • the undrawn yarn 36 is supplied into hot water 38 of a drawing bath 37 and subjected to the first-stage drawing, whereby a yarn roll 39 of the first-stage drawn yarn is obtained.
  • the drawing depends on a speed ratio between a supply nip roller 41 and a take-up nip roller 42 .
  • the first-stage drawn yarn is unwound from the yarn roll 39 , supplied to a dry-heat drawing machine 43 , and subjected to the second-stage drawing inside a guide 47 .
  • the drawing depends on a speed ratio between a supply nip roller 45 and a take-up nip roller 46 .
  • the drawn yarn is wound as a yarn roll 44 .
  • an artificial polypeptide fiber is obtained.
  • the obtained artificial polypeptide fiber has a stress of 350 MPa or more and a toughness of 138 MJ/m 3 or more.
  • the toughness is calculated from an integral value of a stress-strain curve (SS curve) at the time of measuring the strength elongation of the fiber.
  • FIG. 3 illustrates the toughness of the fiber obtained in one example of the present invention, and shows a stress-displacement (strain) curve and toughness (shaded part). This indicates that, when the stress and rupture elongation are both high, the toughness also is high.
  • the stress of the artificial polypeptide fiber of the present invention is preferably 400 MPa or more, more preferably 590 MPa or more, and particularly preferably 620 MPa or more.
  • the rupture elongation is preferably 39% or more, more preferably 45% or more, and further preferably 50% or more.
  • the toughness is preferably 170 MJ/m 3 or more, more preferably 240 MJ/m 3 or more, and further preferably 260 MJ/m 3 or more.
  • An initial elastic modulus of the artificial polypeptide fiber of the present invention is preferably 8 GPa or more, more preferably 14 GPa or more, and further preferably 16 GPa or more.
  • the diameter of the artificial polypeptide fiber of the present invention preferably ranges from 5 to 100 ⁇ m. This range allows the stable supply of drawn yarns.
  • the fiber diameter more preferably ranges from 7 to 30 ⁇ m, and further preferably ranges from 8 to 25 ⁇ m.
  • it is preferable that the diameter of the artificial polypeptide fiber of the present invention is uniform and the variation of the fiber diameter is 5% or less.
  • the fineness unit: tex or deci tex
  • the cross section is not limited to the round shape and may have various shapes. Therefore, the fiber diameter (average diameter) as used herein refers to an average diameter based on the assumption that the cross section is round.
  • the polypeptide preferably is a polypeptide derived from ADF3 that is one of two major dragline proteins of Araneus diadematus. This polypeptide has advantages of basically having high strength elongation and toughness and being synthesized easily.
  • a birefringence ⁇ n ( ⁇ 1000) of the artificial polypeptide fiber preferably is 15.6 or more.
  • the birefringence and a birefringence degree are the same.
  • the birefringence can be measured using a compensator called Senarmont, which is a polarizing microscope manufactured by Olympus Corporation. The measurement range is 0 to 546 nm (0 to 1 ⁇ ).
  • the birefringence ⁇ n ( ⁇ 1000) of 15.6 or more indicates that the orientation of molecules is progressing.
  • chemical cross-links may be formed between polypeptide molecules in a fibroin fiber.
  • functional groups that can be used for cross-links in the polypeptide include amino groups, carboxyl groups, thiol groups and hydroxy groups, but they are not limited to these.
  • An amino group of a lysine side chain contained in the polypeptide can be cross-linked with a carboxyl group of a glutamic acid or an aspartic acid side chain by amide bonds by dehydration condensation.
  • Cross-links may be formed by a dehydration condensation reaction under vacuum heating, or by a dehydration condensation agent such as carbodiimide.
  • cross-linking agent such as glutaraldehyde also may be used.
  • an enzyme such as transglutaminase may be used to form cross-links.
  • the cross-linking reaction may be caused using the cross-linking agent such as carbodiimide, glutaraldehyde, etc.
  • Carbodiimide is represented by the general formula: R 1 N ⁇ C ⁇ NR 2 (where R 1 and R 2 indicate an organic group containing an alkyl group with a carbon number of 1 to 6, or a cycloalkyl group), and specific compounds thereof include 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N,N′-dicyclohexylcarbodiimide (DCC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide, diisopropyl carbodiimide (DIC), etc.
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • DCC N,N′-dicyclohexylcarbodiimide
  • DIC diisopropyl carbodiimide
  • a cross-linking treatment is performed preferably by applying the cross-linking agent to drawn yarns and forming cross-links by vacuum heating-drying.
  • the cross-linking agent may be applied to fibers in a pure form or may be diluted to the concentration of 0.005 to 10 mass % using a lower alcohol with a carbon number of 1 to 5, a buffer solution, etc.
  • the temperature is 20 to 45° C. and the time is 3 to 42 hours.
  • the cross-linking treatment using the cross-linking agent allows artificial polypeptide drawn fibers to have further high stress (strength).
  • ADF3 Part of the amino acid sequence of ADF3 (GI: 1263287), which is one of two major dragline proteins of Araneus diadematus, was obtained from the NCBI web database, and synthesis of a gene encoding an amino acid sequence (SEQ ID NO: 5) was outsourced to GenScript, Inc.
  • the amino acid sequence (SEQ ID NO: 5) is one obtained by adding an amino acid sequence (SEQ ID NO: 7) composed of a start codon at a N-terminal, His 10 tags and an HRV3C Protease (Human rhinovirus 3C Protease) recognition site, to the said amino acid sequence of ADF3.
  • a pUC57 vector to which a gene of ADF3Kai having a base sequence represented by SEQ ID NO: 8 had been introduced was obtained (having an Nde I site immediately upstream of 5′ terminal of gene and an Xba I site immediately downstream of 5′ terminal of gene). Then, the gene was subjected to a restriction enzyme treatment with Nde I and EcoR I, and recombined into a pET22b(+) expression vector.
  • ADF3Kai used as a template, the PCR reaction was performed using a T7 promoter primer (SEQ ID NO: 11) and a Rep Xba I primer (SEQ ID NO: 12).
  • the half of the genome sequence of ADF3Kai on the 5′ side (hereinafter, referred to as a sequence A) was amplified, and the fragment was recombined into a pUC118 vector that in advance had been subjected to the restriction enzyme treatment with Nde I and Xba I using a Mighty Cloning Kit (manufactured by TAKARA BIO INC.).
  • the PCR reaction was performed using an Xba I Rep primer (SEQ ID NO: 13) and a T7 terminator primer (SEQ ID NO: 14).
  • SEQ ID NO: 13 The half of the genome sequence of ADF3Kai on the 3′ side (hereinafter, referred to as a sequence B) was amplified, and the fragment was recombined into a pUC118 vector that in advance had been subjected to the restriction enzyme treatment with Xba I and EcoR I using the Mighty Cloning Kit (manufactured by TAKARA BIO INC.).
  • the pUC118 vector to which the sequence A had been introduced and the pUC118 vector to which the sequence B had been introduced were subjected to the restriction enzyme treatment with Nde I, Xba I and Xba I, EcoR I, respectively, and target DNA fragments of the sequences A and B were purified by gel cut.
  • the DNA fragments A, B and the pET22b(+) that in advance had been subjected to the restriction enzyme treatment with Nde I and EcoR I were subjected to a ligation reaction and transformed into Escherichia coli DH5 ⁇ .
  • plasmid was extracted from a colony where a target band size (3.6 kbp) was obtained, and the entire base sequence was checked by a sequence reaction using a 3130 ⁇ 1 Genetic Analyzer (Applied Biosystems). Consequently, the construction of a gene of ADF3Kai-Large represented by SEQ ID NO: 9 was confirmed.
  • the amino acid sequence of ADF3Kai-Large was as represented by SEQ ID NO: 6.
  • the pET22b(+) expression vector containing the genome sequence of ADF3Kai-Large-NRSH1 obtained above was transformed into Escherichia coli Rosetta (DE3).
  • the obtained single colony was grown for 15 hours in an LB culture medium (2 mL) containing ampicillin.
  • the culture solution (1.4 ml) was added to an LB culture medium (140 mL) containing ampicillin, and grown to an OD 600 of 3.5 under conditions of 37° C. and 200 rpm.
  • the culture solution with the OD 600 of 3.5 was added together with 50% glucose (140 mL) to a 2 ⁇ YT culture medium (7 L) containing ampicillin, and grown further to the OD 600 of 4.0.
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • the bacterial cells collected two hours after the addition of IPTG were washed in a 20 mM Tris-HCl buffer (pH 7.4).
  • the bacterial cells after washing were suspended in a 20 mM Tris-HCl buffer solution (pH 7.4) containing PMSF (about 1 mM) and broken with a high-pressure homogenizer (GEA Niro Soavi).
  • the broken cells were centrifuged and a precipitate was obtained.
  • the obtained precipitate was washed in a 20 mM Tris-HCL buffer solution (pH 7.4) until it achieved high purity.
  • the precipitate after washing was dissolved in a 7.5 M Urea DB buffer solution (7.5 M urea, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, mM Tris-HCl, pH 7.0) and stirred with a stirrer, and then was dialyzed in water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.).
  • White aggregate protein obtained after dialysis was collected by centrifugation, water was removed by a freeze dryer, and freeze-dried powder was collected.
  • a degree of purification of the target protein (about 101.1 kDa) in the obtained freeze-dried powder was checked by performing an image analysis of the results of polyacrylamide gel powder electrophoresis using Totallab (nonlinear dynamics Ltd.). As a result, the degree of purification of ADF3Kai-Large-NRSH1 was about 85%.
  • Hexafluoroisopropanol was added to the freeze-dried powder so that the concentration of the freeze-dried powder became 8.1 mass %. After 14 hours of dissolution using a rotator, dusts and bubbles were removed. The viscosity of the solution was 1,200 cp (centipoises). The obtained solution was defined as the spinning solution (dope solution).
  • the method shown in FIGS. 2A and 2B was adopted as the spinning process and the drawing process.
  • the spinning solution was filled in a cylinder and extruded into 100 mass % methanol coagulation liquid from a nozzle 0.2 mm in diameter using a syringe pump. Thus, undrawn yarns were produced.
  • the extrusion speed was set at 1.8 ml/h, and the length of the coagulation liquid bath was 400 mm.
  • the undrawn yarns were drawn to 2.3 to 7 times in hot water at 80° C.
  • the winding speed was 2.3 to 3.6 m/min.
  • Table 2 summaries various properties of the fibers.
  • the stress-displacement (strain) curves of the fibers obtained in Examples and Comparative Examples are shown in FIGS. 3-9 , respectively.
  • FIG. 7 Comp. Ex. 1 174.7 1.3 5.0 71.3 31 0.77 0.00148 98.1
  • FIG. 8 Comp. Ex. 2 174.8 1.3 5.8 62.6 25 0.61 0.00095 96.8 FIG. 9
  • products of Examples of the present invention are high-toughness artificial polypeptide fibers having favorable stress and rupture elongation.
  • Table 3 summaries the stress, the elongation and the toughness of the fibers obtained in Examples 1-5 of the present invention, and of a conventional natural spider silk fiber, a carbon fiber and a para-based aramid fiber.
  • the artificial polypeptide fiber of the present invention can be used suitably as reinforcing fibers of resin and metal, a composite material, an injection molding, etc.
  • the uses can be applied to a transport machine member such as a car, and a reinforcing fiber of a tire.
  • a fishing line strings of tennis and badminton rackets, a string of a violin, a bowstring of a violin, and artificial hair. It can be in a form of a yarn, a cotton, a weave, a knit, a braid, a nonwoven fabric, etc.

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WO2012165476A1 (fr) 2012-12-06
US20140058066A1 (en) 2014-02-27
JP2014129639A (ja) 2014-07-10
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JP5540154B2 (ja) 2014-07-02
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